1,25(OH)2 vitamin D3 (1,25D) and the vitamin D3 receptor (VDR) regulate cell cycle, differentiation, and apoptosis in the normal mammary gland and in breast cancer cells. The enzyme that catalyzes the production of 1,25(OH)2D3 is the 25-hydroxyvitamin D [25(OH)D3] 1alpha-hydroxylase (1-OHase). Although initially thought to be exclusively expressed in kidney, we have demonstrated expression of 1-OHase in human mammary epithelial cells and in normal murine mammary gland. These studies will test the hypothesis that normal breast tissue expresses 1-OHase and generates 1,25(OH)2D3 which activates VDR locally to inhibit growth and maintain differentiation of mammary epithelial cells. This hypothesis suggests that vitamin D metabolizing enzymes and the VDR play key roles in nutritional regulation of gene expression in mammary gland. A corollary to this hypothesis is that deregulation of 1,25(OH)2D3 production or action at the tissue level may be associated with neoplastic progression of the breast. The following specific aims will test this hypothesis and its implications: Aim 1. To demonstrate a functional role for the 25(OH) vitamin D 1-OHase in mammary gland; Aim 2. To examine metabolism, uptake and transport of vitamin D steroids in mammary epithelial cells; Aim 3. To determine whether non-genomic signaling contributes to the effects of VDR on breast cancer cell growth. Our approaches will include both in vitro and in vivo model systems including both normal and transformed cell lines derived from mice with targeted deletion of 1-OHase or VDR. These studies will provide mechanistic detail on the functions of the vitamin D signaling pathway in mammary gland in relation to prevention and treatment of breast cancer.